1. Field of the Invention
The present invention relates to flow cytometers, and more specifically, to an ultra-miniature personal flow cytometer (pFCM) for the measurement of laser-induced fluorescence through microchip optical transport technology.
2. Description of Related Art
Optically based instrumentation is the mainstay of analytical and industrial-process equipment. Several analytical techniques have evolved from the original first generation large optical components to second generation microchip-based components. However, there remains certain technology areas, including flow cytometry, where both established and new applications lack microchip-based instrumentation, due to the absence of a microengineered approach to the specific optical requirements.
Flow cytometry is a powerful technique for discovering the optical characteristics of microscopic biological particles (e.g., cells or organelles), with widespread applications including immunology, genetics, microbiology and oncology. Optical flow cytometers use optical parameters such as light scatter and fluorescence to detect physical and chemical properties of the particles. For measurement, particles are arranged in single file, typically by hydrodynamic focussing within a sheath fluid, and are interrogated by a light beam (usually laser) as they flow through the beam. Every cell passing through the laser beam scatters light so that light scatter can be used in conjunction with fluorescence probes to discriminate between stained and unstained cells within a sample. Light scatter is sensitive to cell size, shape, and refractive indices of the plasma membrane and internal structures within the cell. Fixatives and stains change refractive indices and thus affect light scatter. The sensitivity of light scatter to all these parameters is dependent on the scattering angle, the shape of the illuminating beam, and the solid angle over which the light is collected. Scattered light is measured in a near forward direction by a photodetector. In addition, a second photodetector is often positioned at 90.degree. to the forward scattering direction to collect large angle light scatter and fluorescence emitted by the particles.
Fluorescence is light remitted following absorption to light energy. Although a wide range of organic molecules found within cells are capable of fluorescence, flow cytometry generally depends on the addition of specific fluorescent dye molecules. Common fluorescence techniques rely on the dye binding specifically to some cellular component, such as DNA, which can then be measured by the fluorescence signal. Thermodynamics requires that emitted light be at a lower energy level than the exciting light that induces the fluorescence. Therefore, fluorescence must be at a longer wavelength, than the exciting light, and this separation in wavelength is known as the Stokes shift. The Stokes shift enables the exciting and emitted light to be separated by optical filters so that the fluorescence can be measured in spite of a huge background of exciting light. The magnitude of the Stokes shift varies with different fluorescent molecules, and it is therefore possible to separate the fluorescence emitted by different molecules excited by the same light source. The simultaneous measurement of (several) fluorescent compounds, together with light scattering measurements (at one or more angles) on single cells illustrates the potential of flow cytometry for multi-parameter data.
Generally, lenses are used to collect fluorescence to transmit it to the photodetectors. Light shielding techniques, including placing apertures in the image plane of the collection lens, are employed to prevent most of the forward scattered light and stray light from reaching the fluorescence photodetector(s). Nevertheless, the lens will also collect some scattered light and stray light. Therefore, optics which limit the bandwidth of light reaching the photodetector are employed. This is often accomplished by optical filters that either absorb or reflect light outside the spectral region of interest. The photodetector used to measure the forward scatter is typically a photodiode, such as a solar cell, while the fluorescence detector (90.degree. scatter) is generally a photomultiplier tube (PMT) because of the higher gain available with these tubes.
Prior art flow cytometers are much larger and more complicated than the pFCM of the present invention. This size translates to cost and complexity. The units generally take up a large amount of bench real estate and are relatively delicate. Thus, there exists a need for a miniaturized flow cytometry system for laboratory use that provides portability, simplicity, increased efficiency, and reduced cost.
It is therefore an object of this invention to provide a miniature pFCM for use in analyzing cell structures.
It is another object of this invention to provide microchip optical transport technology for use in a pFCM.
It is still a further object of this invention to provide a miniature optical module consisting of several components including a microchip demultiplexing waveguide to separate visible fluorescent wavelengths.